Cyclin Dependent Kinases (Cdks) control the many processes of cell cycle progression including DNA replication. In the fission yeast Schizosaccharomyces pombe, Cdc2, the fission yeast Cdk, binds to the DNA replication initiation factors Orp2 and Cdc18. Experiments are proposed to learn how interaction between Cdc2 and the replication initiation factor Orp2 influences the regulation of DNA replication in vivo. First, Cdc2-Orp2 binding will be characterized in vitro; then assays will be developed to detect Cdc2 at replication complexes in vivo. To determine biological significance of Cdc2 at replication origins, Orp2 binding defective mutants of Cdc2 will be isolated and studied in vivo. The mutants will be characterized to identify those that are specifically defective for the Cdc2--Orp2 interaction but are otherwise full length and properly folded. These experiments address the interaction of a natural substrate, Orp2, with a Cdk -- this may show more generally how Cdks recognize substrates. Since Cdc2 acts both positively to initiate replication and negatively to prevent re-replication, it will be exciting to see what phenotypes result from disrupting the interaction between Cdc2 and an initiation factor. Fission yeast has been one of the most important model systems for studies of the cell cycle and there is a wide range of molecular genetic tools with which to determine exactly what is happening to DNA replication and cell cycle progression in Cdc2 mutants. Studies on cell cycle and DNA replication initiation are relevant to cancer in several ways. 1) Fuller understanding of DNA replication may lead to more sophisticated anti-replication drugs. Targeting replication initiation may be a way to take advantage of the lack of checkpoints in many cancer cells. 2) Cancer is a disease of an aberrant cell cycle, and the cell cycle is globally regulated by Cdk kinases. Experiments proposed will elucidate Cdk function in general through a better understanding of its interaction with a natural substrate. 3) Finally, these studies may shed light on the once per cell cycle control of DNA replication which is critical for genome stability.